[Show abstract][Hide abstract] ABSTRACT: The heterotrimeric laminins are a defining component of basement membranes and essential for tissue formation and function in all animals. The three short arms of the cross-shaped laminin molecule are composed of one chain each and their tips mediate the formation of a polymeric network. The structural basis for laminin polymerisation is unknown. We have determined crystal structures of the short-arm tips of the mouse laminin β1 and γ1 chains, which are grossly similar to the previously determined structure of the corresponding α5 chain region. The short-arm tips consist of a laminin N-terminal (LN) domain that is attached like the head of a flower to a rod-like stem formed by tandem laminin-type epidermal growth factor-like (LE) domains. The LN domain is a β-sandwich with elaborate loop regions that differ between chains. The γ1 LN domain uniquely contains a calcium binding site. The LE domains have little regular structure and are stabilised by cysteines that are disulphide-linked 1-3, 2-4, 5-6 and 7-8 in all chains. The LN surface is not conserved across the α, β and γ chains, but within each chain subfamily there is a striking concentration of conserved residues on one face of the β-sandwich, while the opposite face invariably is shielded by glycans. We propose that the extensive conserved patches on the β and γ LN domains mediate the binding of these two chains to each other, and that the α chain LN domain subsequently binds to the composite β-γ surface. Mutations in the laminin β2 LN domain causing Pierson syndrome are likely to impair the folding of the β2 chain or its ability to form network interactions.
[Show abstract][Hide abstract] ABSTRACT: The polymerization of laminin into a cell-associated network--a key step in basement membrane assembly--is mediated by the laminin amino-terminal (LN) domains at the tips of the three short arms of the laminin αβγ-heterotrimer. The crystal structure of a laminin α5LN-LE1-2 fragment shows that the LN domain is a β-jelly roll with several elaborate insertions that is attached like a flower head to the stalk-like laminin-type epidermal growth factor-like tandem. A surface loop that is strictly conserved in the LN domains of all α-short arms is required for stable ternary association with the β- and γ-short arms in the laminin network.
[Show abstract][Hide abstract] ABSTRACT: Dystroglycan is a ubiquitously expressed cell adhesion protein. Its principal role has been determined as a component of the dystrophin-glycoprotein complex of muscle, where it constitutes a key component of the costameric cell adhesion system. To investigate more fundamental aspects of dystroglycan function in cell adhesion, we examined the role of dystroglycan in the dynamics and assembly of cellular adhesions in myoblasts. We show that beta-dystroglycan is recruited to adhesion structures and, based on staining for vinculin, that overexpression or depletion of dystroglycan affects both size and number of fibrillar adhesions. Knockdown of dystroglycan increases the size and number of adhesions, whereas overexpression decreases the number of adhesions. Dystroglycan knockdown or overexpression affects the ability of cells to adhere to different substrates, and has effects on cell migration that are consistent with effects on the formation of fibrillar adhesions. Using an SH3 domain proteomic screen, we identified vinexin as a binding partner for dystroglycan. Furthermore, we show that dystroglycan can interact indirectly with vinculin by binding to the vinculin-binding protein vinexin, and that this interaction has a role in dystroglycan-mediated cell adhesion and spreading. For the first time, we also demonstrate unequivocally that beta-dystroglycan is a resident of focal adhesions.
Full-text · Article · Jan 2010 · Journal of Cell Science
[Show abstract][Hide abstract] ABSTRACT: Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.
[Show abstract][Hide abstract] ABSTRACT: The laminin G-like (LG) domains of laminin-111, a glycoprotein widely expressed during embryogenesis, provide cell anchoring
and receptor binding sites that are involved in basement membrane assembly and cell signaling. We now report the crystal structure
of the laminin α1LG4-5 domains and provide a mutational analysis of heparin, α-dystroglycan, and galactosylsulfatide binding.
The two domains of α1LG4-5 are arranged in a V-shaped fashion similar to that observed with laminin α2 LG4-5 but with a substantially
different interdomain angle. Recombinant α1LG4-5 binding to heparin, α-dystroglycan, and sulfatides was dependent upon both
shared and unique contributions from basic residues distributed in several clusters on the surface of LG4. For heparin, the
greatest contribution was detected from two clusters, 2719RKR and 2791KRK. Binding to α-dystroglycan was particularly dependent on basic residues within 2719RKR, 2831RAR, and 2858KDR. Binding to galactosylsulfatide was most affected by mutations in 2831RAR and 2766KGRTK but not in 2719RKR. The combined analysis of structure and activities reveal differences in LG domain interactions that should enable dissection
of biological roles of different laminin ligands.
Preview · Article · May 2007 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Slit is a large secreted protein that provides important guidance cues in the developing nervous system and in other organs.
Signaling by Slit requires two receptors, Robo transmembrane proteins and heparan sulfate (HS) proteoglycans. How HS controls
Slit-Robo signaling is unclear. Here we show that the second leucine-rich repeat domain (D2) of Slit, which mediates binding
to Robo receptors, also contains a functionally important binding site for heparin, a highly sulfated variant of HS. Heparin
markedly enhances the affinity of the Slit-Robo interaction in a solid-phase binding assay. Analytical gel filtration chromatography
demonstrates that Slit D2 associates with a soluble Robo fragment and a heparin-derived oligosaccharide to form a ternary
complex. Retinal growth cone collapse triggered by Slit D2 requires cell surface HS or exogenously added heparin. Mutation
of conserved basic residues in the C-terminal cap region of Slit D2 reduces heparin binding and abolishes biological activity.
We conclude that heparin/HS is an integral component of the minimal Slit-Robo signaling complex and serves to stabilize the
relatively weak Slit-Robo interaction.
Full-text · Article · Jan 2007 · Journal of Biological Chemistry